Polydatin which is known as stilbenoid-based drug has effects on anti-oxidant, anti-aging, anti-virus, neuroprotective action, and anti-inflammatory action. Polydatin is a stilbenoid glucoside, and is a derivative of resveratrol, and the stilbenoid-based drug such as polydatin is known to be metabolized by CYP1A2 and CYP1B1 which are cytochrome P450 enzymes present in human liver. However, metabolic pathways and metabolites of polydatin have not been specifically known so far.
The cytochrome P450 enzyme is present throughout all living organisms including archaea, bacteria, fungi, plants, animals, and human, and corresponds to a large family consisting of enzymes serving as catalysts promoting various oxidation reaction (http://drnelson.uthsc.edu/CytochromeP450.html).
P450 is significantly various in view of kinds, and may function as catalysts for a wide range of substrates, thereby having a potential for being effectively useful as a biological catalyst in production of fine chemicals such as pharmaceutical products. However, despite the potential usefulness, P450 enzymes have problems in being used as a biological catalyst due to low stability, catalytic activity, and availability.
P450 is capable of being used to synthesize drug metabolites, in particular, chiral metabolites by a simple, highly efficient, and environment-friendly method, wherein the synthesis of the chiral metabolites is not suitably performed by chemical methods, but needs to use an enzyme. If the drug acts as a prodrug, the drug is converted to a biologically active metabolite by P450s of the human liver when the drug metabolizes after administration. Here, in order to conduct research into drug efficacy, toxic, pharmacokinetics, and the like, of active metabolites produced from the prodrugs, a large amount of pure metabolites are needed. Further, when the metabolites themselves have biological activities, direct administration of the metabolites in vivo may provide big benefits, such that technique for mass-producing the metabolites is significantly important.
However, there are a lot of problems in chemically synthesizing pure metabolites. In addition, as an alternative thereof, a human P450 enzyme is used, but it is technically almost impossible to directly separate only P450 enzymes from a human sample. Further, even in the case of animal-derived P450, it is difficult to perform pure-separation, and enzyme activity is also low. In order to solve the problems, there was an attempt to produce metabolites using human P450 which is heterologously expressed in E. coli or insect cells, but the attempt has problems such as limited stability, a slow response rate, high cost and low productivity.
As another method, there was a method using bacterial P450 enzymes which are engineered to have catalytic activity, and a possibility that P450 enzymes capable of functioning on various substrates are appropriately usable for producing biotechnological metabolites by using the engineered enzymes has been reported.
Therefore, the present inventors conducted research into pathways and metabolites of polydatin, found bacterial P450 chimera enzyme capable of selectively producing specific metabolites among the metabolites of polydatin, and completed the present invention.